Stem Cell Reviews and Reports

, Volume 11, Issue 1, pp 39–49 | Cite as

MicroRNA-221 is Required for Proliferation of Mouse Embryonic Stem Cells via P57 Targeting

  • Jin Li
  • Yihua Bei
  • Qi Liu
  • Dongchao Lv
  • Tianzhao Xu
  • Yanyun He
  • Ping Chen
  • Junjie XiaoEmail author


Factors responsible for the rapid proliferative properties of embryonic stem (ES) cells are largely unknown. MicroRNA-221/222 (miR-221/222) regulate proliferation in many somatic cells, however, their roles in proliferation of ES cells are unclear. In this study, E14 mouse ES cells proliferation was determined by total cell counting, Cell Counting Kit (CCK-8), size of colonies and cell cycle analysis, while apoptosis and necrosis using Annexin V and propidium iodide staining. miR-221 inhibitor decreased proliferation of ES cells without inducing apoptosis and necrosis. miR-221 mimic, miR-222 mimic and miR-222 inhibitor did not affect ES cells proliferation. The expression level of miR-221 remained unchanged upon embryoid body (EB) formation. ES cells with miR-221 inhibition maintained an undifferentiated state, as indicated by unchanged alkaline phosphatase enzyme activity and Sox2, Nanong, and Oct4 expressions. P57 was post-transcriptionally regulated by miR-221 in ES cells. P57 knockdown completely abolished the inhibition effects of ES cells proliferation observed in miR-221 reduction, further indicating that miR-221 inhibition is likely to mediate its antiproliferative effects via P57 expression. To exclude that the function of miR-221 in ES cells is E14 specific, the effects of miR-221 mimic and inhibitor in size of colonies and cell cycle of R1 mouse ES cells were also determined and similar effects in inhibiting proliferation were achieved with miR-221 inhibition. Therefore, miR-221 is required for mouse ES cells proliferation via P57 targeting. This study indicates that miR-221 is among the regulators that control ES cells proliferation and might be used to influence the fate of ES cells.


Mouse embryonic stem cells Microrna-221 Microrna-222 Proliferation P57 



This work was supported by the grants from National Natural Science Foundation of China (81200169 to J. Xiao), Innovation Program of Shanghai Municipal Education Commission (13YZ014 to J. Xiao), Foundation for University Young Teachers by Shanghai Municipal Education Commission (year 2012, to J. Xiao), Innovation fund from Shanghai University (sdcx2012038 to J. Xiao), and Program for the integration of production, teaching and research for University Teachers supported by Shanghai Municipal Education Commission (year 2014, to J. Xiao).

Conflict of Interest

The authors declare there are no conflicts of interest.

Author Contributions

Jin Li: Collection of data, and data analysis.

Yihua Bei: Collection of data, data analysis, and manuscript writing.

Qi Liu: Data analysis, and manuscript writing.

Dongchao Lv: Collection of data.

Tianzhao Xu: Collection of data.

Yanyun He: Collection of data.

Ping Chen: Data analysis.

Junjie Xiao: Concept and design, financial support, data anysis, manuscript writing, final approval of manuscript.


  1. 1.
    Yang, A., Shi, G., Zhou, C., et al. (2011). Nucleolin maintains embryonic stem cell self-renewal by suppression of p53 protein-dependent pathway. The Journal of Biological Chemistry, 286, 43370–43382.CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Ho, L., Ronan, J. L., Wu, J., et al. (2009). An embryonic stem cell chromatin remodeling complex, esBAF, is essential for embryonic stem cell self-renewal and pluripotency. Proceedings of the National Academy of Sciencesof the United States of. America, 106, 5181–5186.Google Scholar
  3. 3.
    Wang, Y., & Blelloch, R. (2009). Cell cycle regulation by MicroRNAs in embryonic stem cells. Cancer Research, 69, 4093–4096.CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Nishii, T., Oikawa, Y., Ishida, Y., Kawaichi, M., & Matsuda, E. (2012). CtBP-interacting BTB zinc finger protein (CIBZ) promotes proliferation and G1/S transition in embryonic stem cells via Nanog. The Journal of Biological Chemistry, 287, 12417–12424.CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Heo, J. S., Lee, Y. J., & Han, H. J. (2006). EGF stimulates proliferation of mouse embryonic stem cells: involvement of Ca2+ influx and p44/42 MAPKs. The American Journal of Physiology- Cell Physiology, 290, 123–133.CrossRefGoogle Scholar
  6. 6.
    Rodriguez-Gomez, J. A., Levitsky, K. L., & Lopez-Barneo, J. (2012). T-type Ca2+ channels in mouse embryonic stem cells: modulation during cell cycle and contribution to self-renewal. The American Journal of Physiology- Cell Physiology, 302, C494–504.CrossRefGoogle Scholar
  7. 7.
    Raz, R., Lee, C. K., Cannizzaro, L. A., D'Eustachio, P., Levy, D. E., & Levy, D. E. (1999). Essential role of STAT3 for embryonic stem cell pluripotency. Proceedings of the National Academy of Sciencesof the United States of America, 96, 2846–2851.CrossRefGoogle Scholar
  8. 8.
    Ma, T., Wang, Z., Guo, Y., & Pei, D. (2009). The C-terminal pentapeptide of nanog tryptophan repeat domain interacts with Nac1 and regulates stem cell proliferation but not pluripotency. The Journal of Biological Chemistry, 284, 16071–16081.CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Xu, J., Zhao, J., Evan, G., Xiao, C., Cheng, Y., & Xiao, J. (2012). Circulating microRNAs: novel biomarkers for cardiovascular diseases. Journal of Molecular Medicine, 90, 865–875.CrossRefPubMedGoogle Scholar
  10. 10.
    Seeger, F. H., Zeiher, A. M., & Dimmeler, S. (2013). MicroRNAs in stem cell function and regenerative therapy of the heart. Arteriosclerosis, Thrombosis, and Vascular Biology, 33, 1739–1746.CrossRefPubMedGoogle Scholar
  11. 11.
    Li, J., Xu, J., Cheng, Y., Wang, F., Song, Y., & Xiao, J. (2013). Circulating microRNAs as mirrors of acute coronary syndromes: MiRacle or quagMire? Journal of Cellular and Molecular Medicine, 17, 1363–1370.CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Mayoral, R. J., Deho, L., Rusca, N., Bartonicek, N., Saini, H. K., Enright, A. J., & Monticelli, S. (2011). MiR-221 influences effector functions and actin cytoskeleton in mast cells. PloS One, 6, e26133.CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Xiao, J., Liang, D., Zhang, H., et al. (2012). MicroRNA-204 is required for differentiation of human-derived cardiomyocyte progenitor cells. Journal of Molecular and Cellular Cardiology, 53, 751–759.CrossRefPubMedGoogle Scholar
  14. 14.
    Xiao, J., Liang, D., Zhang, Y., et al. (2011). MicroRNA expression signature in atrial fibrillation with mitral stenosis. Physiological Genomics, 43, 655–664.CrossRefPubMedGoogle Scholar
  15. 15.
    Wong, Q. W. L., Ching, A. K. K., Chan, A. W. H., et al. (2010). MiR-222 overexpression confers cell migratory advantages in hepatocellular carcinoma through enhancing AKT signaling. Clinical Cancer Research, 16, 867–875.CrossRefPubMedGoogle Scholar
  16. 16.
    Pineau, P., Volinia, S., McJunkin, K., et al. (2010). miR-221 overexpression contributes to liver tumorigenesis. Proceedings of the national academy of sciencesof the united states of. America, 107, 264–269.Google Scholar
  17. 17.
    Tan, L., Yu, J. T., & Hu, N. (2013). Non-coding RNAs in Alzheimer’s disease. Molecular Neurobiology, 47, 382–393.CrossRefPubMedGoogle Scholar
  18. 18.
    Chen, S. L., Zheng, M. H., Shi, K. Q., Yang, T., & Chen, Y. P. (2013). A new strategy for treatment of liver fibrosis: letting MicroRNAs do the job. BioDrugs, 27, 25–34.CrossRefPubMedGoogle Scholar
  19. 19.
    Liu, X., Cheng, Y., Zhang, S., Lin, Y., Yang, J., & Zhang, C. (2009). A necessary role of miR-221 and miR-222 in vascular smooth muscle cell proliferation and neointimal hyperplasia. Circulation Research, 104, 476–487.CrossRefPubMedCentralPubMedGoogle Scholar
  20. 20.
    Liu, X., Cheng, Y., Yang, J., Xu, L., & Zhang, C. (2012). Cell-specific effects of miR-221/222 in vessels: molecular mechanism and therapeutic application. Journal of Molecular and Cellular Cardiology, 52, 245–255.CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Sage, C., Nagel, R., Egan, D. A., et al. (2007). Regulation of the p27 (Kip1) tumor suppressor by miR-221 and miR-222 promotes cancer cell proliferation. EMBO Journal, 26, 3699–3708.CrossRefPubMedCentralPubMedGoogle Scholar
  22. 22.
    Fornari, F., Gramantieri, L., Ferracin, M., et al. (2008). MiR-221 controls CDKN1C/p57 and CDKN1B/p27 expression in human hepatocellular carcinoma. Oncogene, 27, 5651–5661.CrossRefPubMedGoogle Scholar
  23. 23.
    Guo, X., Liu, Q., Wang, G., et al. (2013). MicroRNA-29b is a novel mediator of Sox2 function in the regulation of somatic cell reprogramming. Cell Research, 23, 142–156.CrossRefPubMedCentralPubMedGoogle Scholar
  24. 24.
    Chen, D., Farwell, M. A., & Zhang, B. (2010). MicroRNA as a new player in the cell cycle. Journal of Cellular Physiology, 225, 296–301.CrossRefPubMedGoogle Scholar
  25. 25.
    Zhang, J., Han, L., Ge, Y., et al. (2010). miR-221/222 promote malignant progression of glioma through activation of the Akt pathway. International Journal of Oncology, 36, 913–920.PubMedGoogle Scholar
  26. 26.
    Yu, B., Zhou, S., Wang, Y., et al. (2012). miR-221 and miR-222 promote Schwann cell proliferation and migration by targeting LASS2 after sciatic nerve injury. Journal of Cell Science, 125, 2675–2683.CrossRefPubMedGoogle Scholar
  27. 27.
    Radojicic, J., Zaravinos, A., Vrekoussis, T., Kafousi, M., Spandidos, D. A., & Stathopoulos, E. N. (2011). MicroRNA expression analysis in triple-negative (ER, PR and Her2/neu) breast cancer. Cell Cycle, 10, 507–517.CrossRefPubMedGoogle Scholar
  28. 28.
    Tsai, Z. Y., Singh, S., Yu, S. L., et al. (2010). Identification of microRNAs regulated by activin a in human embryonic stem cells. Journal of Cellular Biochemistry, 109, 93–102.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Jin Li
    • 1
    • 2
  • Yihua Bei
    • 1
    • 2
  • Qi Liu
    • 3
  • Dongchao Lv
    • 1
    • 2
  • Tianzhao Xu
    • 1
    • 2
  • Yanyun He
    • 1
  • Ping Chen
    • 1
    • 2
  • Junjie Xiao
    • 1
    • 2
    Email author
  1. 1.Regeneration Lab and Experimental Center of Life Sciences, School of Life ScienceShanghai UniversityShanghaiChina
  2. 2.Innovative Drug Research Center of Shanghai UniversityShanghaiChina
  3. 3.Department of EndocrinologyTongji Hospital, Tongji University School of MedicineShanghaiChina

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